Resistance to TRAIL is mediated by DARPP-32 in gastric cancer.

Abstract

PURPOSE:

Dopamine and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32), is overexpressed during the gastric carcinogenesis cascade. Here, we investigated the role of DARPP-32 in promoting resistance to treatment with TRAIL.

EXPERIMENTAL DESIGN:

In vitro cell models including stable expression and knockdown of DARPP-32 were used. The role of DARPP-32 in regulating TRAIL-dependent apoptosis was evaluated by clonogenic survival assay, Annexin V staining, immunofluorescence, quantitative reverse transcriptase PCR, Western blot, and luciferase reporter assays.

RESULTS:

Stable expression of DARPP-32 in MKN-28 cells enhanced cell survival and suppressed TRAIL-induced cytochrome c release and activation of caspase-8, -9, and -3. Conversely, short hairpin RNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL-induced apoptosis and enhanced TRAIL-mediated activation of caspase-8, -9, and -3. DARPP-32 induced BCL-xL expression through activation of Src/STAT3 signaling, and treatment with the Src-specific inhibitor PP1 abrogated DARPP-32-dependent BCL-xL upregulation and cell survival in MKN-28 cells. The TRAIL treatment induced caspase-dependent cleavage of NF-κBp65 protein; this cleavage was prevented by DARPP-32, thus maintaining NF-κB activity and the expression of its target, FLIP(S) protein. This suggests that upregulation of BCL-xL could play a possible role in blocking the mitochondria intrinsic apoptosis pathway, whereas the DARPP-32 effect on the NF-κB/FLIP(S) axis could serve as an additional negative feedback loop that blocks TRAIL-induced activation of caspase-8.

CONCLUSION:

Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through upregulation of BCL-xL, and the extrinsic apoptosis pathway through the NF-κB/FLIP(S) axis.

• Authors: Abbes Belkhiri, Shoumin Zhu, Zheng Chen, Mohammed Soutto, Wael El-Rifai
• Journal: Clinical cancer research : an official journal of the American Association for Cancer Research (Clin Cancer Res) Vol. 18 Issue 14 Pg. 3889-900 (Jul 15 2012) ISSN: 1557-3265 [Electronic] United States
• PMID: 22589394 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• BCL2L1 protein, human
• CASP8 and FADD-Like Apoptosis Regulating Protein
• Dopamine and cAMP-Regulated Phosphoprotein 32
• NF-kappa B
• PPP1R1B protein, human
• TNF-Related Apoptosis-Inducing Ligand
• TNFSF10 protein, human
• bcl-X Protein
• Caspases

Topics

• Apoptosis (drug effects, genetics)
• CASP8 and FADD-Like Apoptosis Regulating Protein (metabolism)
• Caspases (metabolism)
• Cell Line, Tumor
• Cell Transformation, Neoplastic (drug effects, genetics)
• Dopamine and cAMP-Regulated Phosphoprotein 32 (genetics, metabolism)
• Gene Expression Regulation, Neoplastic (drug effects, genetics)
• Humans
• NF-kappa B (metabolism)
• Signal Transduction
• Stomach Neoplasms (genetics, metabolism)
• TNF-Related Apoptosis-Inducing Ligand (administration & dosage, genetics)
• bcl-X Protein (metabolism)